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Resources for PKD research

A. High-throughput barcoded mutation analysis of all ~1,500 exons of ~90 human renal cystic genes

B. High-throughput barcoded mutation analysis of all ~1,500 exons of ~90 mouse renal cystic genes

C. Whole exome sequencing (WES) of smaller cohorts of human patients

D. Sequencing of gene of interest (candidate PKD modifier or ciliopathy gene) in an existing cohort of PKD-ciliopathy patients

A. Full-length DNA constructs for the expression of normal and mutant PKD proteins (PKD1, PKD2, and PKHD1)

B. Stable cell lines of the full-length PKD1, PKD2, and PKHD1 constructs

C. Monoclonal and polyclonal antibodies against different regions of PC1, PC2 and FPC proteins (if there is a specific domain/type you are intested, please indicate)

D. Kidney tissues, tissue sections, and cystic fluids from normal and ADPKD patients

E. hTERT immortalized renal tubular epithelial cells from ADPKD patients

F. Pkd1 and Pkd2 floxed or germline mutant mice with or without inducible Cre transgene

G. Tissue sections and kidney tissues from wild type and Pkd1 and Pkd2 knockout mice induced at different stages.

H. Primary culture of renal epithelial cells from Pkd1f/f and Pkd2f/f mouse model.

I. Inducible immortalized cell lines from Pkd1f/f and Pkd2f/f mouse model using mouse telomerase reverse transcriptase (mTert).

J. Provide consultations, protocols, and hand-by-hand teaching to investigators who wish to learn kidney cell culture and three-dimensional culture (cystogenesis and tubulogensis assay) using renal tubular epithelial cells.

A. Cellular Signal Transduction: Using cells KD’d, KO’d or mutated for AD/AR-PKD or cystic kidney syndromic genes [Identification of potential signaling defects]

1. Measure steady state activities of cellular signal transduction pathways such as MAP kinase, Hippo, canonical Wnt and Sonic Hedgehog (previously implicated in AD- and ARPKD and ciliopathies) [Immunofluorescence and quantitative immunoblotting and mass

2. Measure the steady-state activity and agonist-induced transcriptional response of jun, yap/taz, tcf and gli transcription factors. [qPCR]

3. Investigate downstream activation of CREB and NFAT transcription factors that are downstream of second messenger signaling of cAMP and Ca2+, respectively [qPCR, Immunofluorescence and quantitative immunoblotting and mass spectrometry]

B. Cellular and Ciliary Dynamics: Using fluorescent protein-based localization and activity reporters [Consultation for detailed temporal measurements of signaling]

1. Measure Hh (Smo translocation, Gli activity), Wnt (Beta-catenin accumulation, TCF actvity) dynamics

2. Measure live cell Ca2+ (ratiometric) and PKA (FRET) dynamics in cytoplasmic and ciliary compartments

3. Measure Intraflagellar transport parameters live cell imaging of IFT and IFT-associated proteins fused to fluorescent proteins

4. Measure DNA damage dynamics, cell division and ciliary membrane protein dynamics (via photoactivation and photobleaching).

C. Subcellular and ciliary organization [Consulation for detailed subcellular imaging]

1. Determine the spatial arrangement of the subcellular organelles (e.g. nucleus, cystoskeleton, golgi apparatus, endoplasmic reticulum, mitochondrial trabeculae, etc.)

2. Investigate protein organization in the primary cilium compartments (e.g. transition zone, basal body, axoneme, etc.) by super-resolution methods (STORM and Structured Illumination)

A. iPS Cell Generation

Distribution of existing iPS cells from patients with ADPKD/ARPKD/ciliopathies
Derivation of new iPS cells from PKD/ciliopathy patient cells (e.g. fibroblasts or kidney epithelial cells)
In vitro characterization of iPS cells to demonstrate pluripotency
Teratoma formation from iPS cells in immunodeficient mice

B. Genome Engineering

Generation of CRISPR constructs for PKD/ciliopathy disease genes
Derivation of mutant iPS cell lines using CRISPRs

C. Directed Differentiation

Differentiation of iPS cells into kidney progenitor cellsDifferentiation of iPS cells into liver progenitor cells
Biological samples (RNA and protein) from iPS cells or derived tissues
Complex organoid formation from iPS cells in vitro
Implantation of iPS-derived somatic cells into liver and kidney of immunodeficient mice
Derivation of mutant somatic cell lines using CRISPRs
Differentiation of iPS cells into endothelial and myocardial progenitor cells
Banking and distribution of iPS cells for other investigators

If you are interested in any resources, please send us a message.

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